Objectives/Goals: The goal of this work is to understand the physiological profile of phage susceptibility and identify candidate phage defense mechanisms. Additionally, it aims to determine the host receptors targeted by bacteriophages to infect E. coli O157:H7 through random bar code transposon-site sequencing (RB-TnSeq). Methods/Study Population: A collection of 109 E. coli O157:H7 strains from environmental, food, and animal sources were analyzed, representing phylogenetic lineages corresponding to clades 2, 3, 5, 6, 7, and 8. Phage susceptibility profiles were determined using 23 bacteriophages, assessing plaque morphology. Using the O157:H7 genomes, a genomic analysis was conducted with the Prokaryotic Antiviral Defense Locator (PADLOC), which identified putative phage defense systems through sequence homology. Additionally, 5 RB-TnSeq libraries were generated in representative strains to study loss-of-function mutations. These libraries will be screened against a subset of diverse phage to identify the receptors involved in phage adsorption. Results/Anticipated Results: The phage resistance patterns showed susceptibility varied across clades, suggesting distinct mechanisms. Several defense systems were identified using PADLOC, including restriction-modification, Cas, Lamassu, and Druantia. Phage defense candidate (PDC) systems were identified, showing homology to known systems, though their specific function remains unknown. Clade 7.2 exhibited higher phage resistance and a greater presence of PDCs compared to the other clades. Five saturated RB-TnSeq libraries were constructed in O157:H7, achieving 84.5–89% gene coverage. These libraries will facilitate the identification of receptors involved in phage adsorption and resistance. Discussion/Significance of Impact: This study deepens our understanding of phage resistance in E. coli O157:H7 by identifying key defense systems and receptors. The discovery of novel antiviral mechanisms offers promising targets for phage-based interventions, potentially enhancing strategies for controlling this dangerous pathogen.

Objectives/Goals: This study will assess population heterogeneity in Candida bloodstream infections by quantifying antifungal resistance, fitness, and genomic diversity to understand clonality and develop a high-throughput screening tool to detect population-level resistance to update clinical practice. Methods/Study Population: This study assesses antifungal resistance and population heterogeneity in Candida bloodstream isolates collected through multiple Midwest hospitals. Blood samples are plated to isolate single colonies and population samples, which are then archived. We test resistance to key antifungals using EUCAST guidelines, conduct growth curve assays, and perform whole-genome sequencing to determine genetic diversity. A high-throughput screening method tracks colony growth under different drug conditions using time-lapse imaging and custom analysis software. The findings will reveal the extent of antifungal resistance and genetic variation within infecting populations, informing better clinical management. Results/Anticipated Results: Preliminary analysis of Candida glabrata bloodstream isolates show significant heterogeneity in colony morphology, antifungal resistance, and fitness. Some single colonies exhibit higher minimum inhibitory concentration values for micafungin and fluconazole than the overall population, while others show reduced susceptibility to amphotericin B, highlighting diverse resistance profiles. Growth assays reveal distinct fitness phenotypes within these populations. This variation underscores the limitations of single-colony testing and suggests a need for population-level resistance screening. We anticipate genomic analyses will identify genetic diversity underlying these differences, supporting a more comprehensive clinical approach to treatment. Discussion/Significance of Impact: This study reveals notable intrapopulation heterogeneity in Candida glabrata, including variations in colony morphology, antifungal resistance, and fitness. Findings highlight genomic diversity, introduce a novel screening method for resistance, and emphasize the need for population-level testing in clinical practice.

Objectives/Goals: To assess theory of mind and empathy in adolescents with Tourette syndrome (TS) and examine their association with social problems. This study aims to extend research in social cognition to an adolescent cohort with TS and identify a potential modifiable risk factor for social problems in TS that may serve as a novel intervention target. Methods/Study Population: We will enroll 50 adolescents with TS (ages 11–17) and 50 demographically matched controls along with one parent to complete a single in-person study visit. Adolescents with TS will be recruited through the Vanderbilt Center for TS and other Tic disorders. Controls will be recruited using university listservs and flyers posted in community and primary care settings. Adolescents will complete the NEPSY-II to assess theory of mind abilities and the Multifaceted Empathy Test – Juvenile to assess empathy with negative emotions. Parents will complete the Child Behavior Checklist to assess adolescent social problems. Results/Anticipated Results: Based on evidence of low self-other distinction in TS, we hypothesize TS adolescents will make more errors about the mental states of others (theory of mind) and report greater emotional reactions to faces (empathy) compared to controls. Further, greater social problems will be associated with greater disturbances in social cognition. To date, 15 adolescents with TS and 15 matched controls have completed the assessment (67% male; Mage  =  14.33 in both groups). Within this sample, adolescents with TS experienced more social problems than controls (Cohen’s d  =  .74, p  = .03). There were no between-group differences in theory of mind or empathy in this pilot sample. However, higher levels of both theory of mind and empathy were linked to experiencing greater social problems in the TS sample only (p’s < .05). Discussion/Significance of Impact: Preliminary findings suggest that while social cognition did not differ between groups, TS adolescents exhibiting high levels of theory of mind and empathy appear to struggle socially. This work could inform future interventions by highlighting the need to focus on social cognition and how these skills translate into social behaviors.

Objectives/Goals: Communication between clinicians and parents of seriously ill infants is understudied. This study aims to 1) define high-quality communication with parents of critically ill infants and 2) evaluate the psychometric properties and validity of a measure of high-quality communication in parents of critically ill infants. Methods/Study Population: 1) Using participant observation and semi-structured interviews of 35 parents of hospitalized infants, I will conduct content analysis to describe high-quality prognostic communication with parents of infants in the pediatric intensive care unit. Using descriptions captured during participant observation and in semi-structured interviews, I will produce a novel definition of high-quality communication with parents of seriously ill infants. I will also explore parent experiences of communication by race. 2) I will validate a measure of communication quality in parents of 200 neonatal and pediatric intensive care unit patients. I will use factor analysis to evaluate the extent to which responses map onto an established construct and assess dimensionality and reliability. Results/Anticipated Results: 1) I anticipate finding that identification of high-quality communication will be consistent between participant observation and interviews and will track with Wreesmann’s framework. I hypothesize that minoritized parents are more likely to receive low-quality communication. 2) I hypothesize that the measure of communication quality will be valid and reliable in the neonatal and pediatric intensive care units. Discussion/Significance of Impact: I will explore communication quality in a novel setting for which limited data are currently available, establishing a measure for future pediatric communication research and identifying targets for interventions to improve communication quality. Better understanding of communication with parents of sick infants will lead to improved outcomes.

Objectives/Goals: Understand the impact of sarcopenia on the main respiratory muscle, the diaphragm (DIAm). We hypothesize that in the DIAm of older (i.e., 24 months) compared to younger (i.e., 6 months) rats, maximum specific force (P0) is reduced, maximum shortening velocity (Vmax) is slower, maximum power output is reduced, and endurance is improved. Methods/Study Population: Mid-costal DIAm strips were excised from 6-month (n = 8; 4 female and 4 male) and 24-month (n = 8; 4 female and 4 male) rats. The DIAm was stimulated using platinum plate electrodes, and mechanical and endurance properties were measured (at 26oC). Results/Anticipated Results: In the DIAm, maximum tetanic formce (P0) decreased by ~35%, maximum velocity of shortening (Vmax) slowed by ~20%, and peak power output was reduced by ~35% in 24-month compared to 6-month rats. During repetitive isovelocity (30% Vmax; approximating peak power output) contractions, endurance (the period during which power output was sustained) of the DIAm was unaffected by aging. Corresponding with previous findings, Discussion/Significance of Impact: The changes in DIAm mechanical performance corresponded to an age-related atrophy of type IIx/IIb muscle fibers. We conclude that force generation and endurance of the DIAm required for breathing motor function is preserved in old age, while DIAm sarcopenia does impair more forceful expulsive airway clearance and voiding behaviors.

Objectives/Goals: Bronchiolitis obliterans syndrome (BOS), a form of chronic lung allograft dysfunction (CLAD) that primarily affects the small airways, is often diagnosed too late using standard pulmonary function tests. This project aims to evaluate whether quantitative air trapping analysis can serve as an early diagnostic tool for BOS. Methods/Study Population: We performed a retrospective analysis of 134 computed tomography scans with inspiratory and expiratory protocols from 73 lung transplant recipients (48 male, 25 female). Quantitative air trapping analysis was performed by VIDA Diagnostics using a supervised machine learning technique called disease probability measure (DPM). Results/Anticipated Results: We found that lung transplant recipients exhibit significantly more air trapping compared to healthy controls and other small airway diseases, such as long COVID and cystic fibrosis. Notably, lung transplant recipients showed increased air trapping in the upper lobes. However, when separating participants into CLAD and non-CLAD groups, those meeting criteria for CLAD had significantly more air trapping in the left lower lobe. Additionally, only 2 out of 16 participants meeting CLAD criteria had less than 20% air trapping in their lungs, suggesting early involvement of the small airways. Discussion/Significance of Impact: Quantitative air trapping analysis seems to be an important diagnostic modality in the early detection of lung transplant-related small airway disease. Prospective longitudinal studies are needed to evaluate the spatial pathophysiology in these patients and to determine whether early air trapping can predict the development of CLAD.

Objectives/Goals: Alzheimer’s disease (AD) has limited treatments and an extremely high rate of clinical trial failure. Through a collaborative effort, Agomelatine (AGO) was identified as having repurposing potential for AD. This study sets out to evaluate the preclinical potential of AGO for the treatment of AD. Methods/Study Population: The TgF344-AD rat model (expresses human mutant “Swedish” amyloid-precursor protein and a Δ exon 9 presenilin 1) was used to test AGO’s potential to reduce cognitive deficits and neuropathology. The model was chosen due to its age-dependent progressive AD pathology and cognitive decline. Treatment with AGO at ~10 mg/kg body weight/day began at 5 months of age (pre-pathology) and continued until 11 months of age when cognitive testing (active place avoidance task) and tissue collection occurred. Immunohistochemistry was used to evaluate amyloid beta plaque burden and microglial response in the hippocampus. Results/Anticipated Results: AGO-treated female TgF344-AD rats showed reduced cognitive deficits with an increased latency to first entrance in aPAT testing compared to nontreated transgenic littermates. There were no differences between the cognitive performance of AGO treated and untreated male TgF344-AD rats. Interestingly, this reduced cognitive deficit did not correlate with decreased amyloid beta pathology in female AGO-treated rats yet male transgenic treated rats did have decreased amyloid burden in the dentate gyrus (DG) of the hippocampus. AGO modulated microglial activation in the DG of female transgenic rats. Discussion/Significance of Impact: AGO reduced cognitive deficits in females, but did not change their amyloid burden. This suggests that AGO could increase resilience to amyloid deposition in female rats. With the recent development of amyloid targeting drugs, novel non-amyloidogenic treatments have a large translational potential.

Objectives/Goals: Our aim was to identify how the epithelial–mesenchymal transition shields heterogeneous breast tumors against immune attack. Additionally, we endeavored to understand whether our findings were conserved in canine mammary tumors as a translational model for human breast tumors. Methods/Study Population: To understand interactions between quasi-mesenchymal (qM) tumor cells, epithelial (E) tumor cells, and immune cells within heterogeneous breast tumors, we utilized a preclinical mouse model established in our lab. In this system, we can precisely control the proportions of E and qM tumor cells within tumors and study what immune cells infiltrate these tumors in response, using flow cytometry and immunofluorescent staining. Using this model, we have also established cell lines to study E and qM tumor cells in vitro. Finally, we used immunohistochemistry to label immune cells in canine mammary tumors and quantified the presence of these cells in relation to the expression of epithelial and mesenchymal cellular markers. Results/Anticipated Results: We observed that immune suppression within heterogeneous mammary tumors is driven by local, rather than systemic, effects of quasi-mesenchymal (qM) tumor cells. The presence of systemic qM-derived factors does not alter immune cell infiltration nor sensitivity to immunotherapy of epithelial (E) tumors. Furthermore, I found that the local activity of qM-derived factors within heterogeneous tumors induces immune-suppressive changes in surrounding E cells, which protects them against immune attack. Finally, I found that canine mammary tumors with higher proportions of qM tumor cells assemble an immune-suppressive tumor microenvironment, highlighting the translational potential of our findings. Discussion/Significance of Impact: We identified that the epithelial–mesenchymal transition induces immune-suppressive changes in heterogeneous tumors. These findings may reveal novel therapeutic targets for treatment of refractory tumors. Our findings in canine tumors suggest that these mechanisms are conserved across species.

Objectives/Goals: Adolescence is a critical period where brain networks are thought to be influenced by environmental factors. This presentation examines violence exposure’s impact on brain connectivity and identifies potential protective factors. Methods/Study Population: A secondary data analysis was conducted using data from a subsample of the Adolescent Brain Cognitive Development Study (release 5.1). Youth who completed victimization questionnaires at two time points were eligible for inclusion, resulting in 2016 participants. Linear regression was utilized to analyze associations between violence exposure measured by the juvenile victimization questionnaire and functional connectivity of specified regions of interests using the Gordon functional parcellation for cortical regions and the Freesurfer parcellation for subcortical regions. Moderation analysis will be utilized to assess the effects of peer and caregiver support on the associations between violence exposure and functional connectivity, currently ongoing. Results/Anticipated Results: Between 18 and 59% of the sample reported experiencing at least one form of violence exposure, with racial differences noted in missing versus complete data. Multiple domains of violence and cumulative exposure were associated with both increased and decreased functional connectivity across within-network, between-network, and network-subcortical regions. At baseline, internet violence was linked to lower within-network connectivity, while peer victimization was associated with higher connectivity at both baseline and follow-up. Between network analysis showed lower connectivity with witnessing violence at baseline and higher connectivity with internet victimization at follow-up. Discussion/Significance of Impact: These findings emphasize the need for further exploration of the underlying mechanisms that link violence exposure to developmental trajectories and identification of protective factors such as caregiver and peer support, to inform interventions and promote resilience in affected youth.

Objectives/Goals: To probe microbe and bacterial extracellular vesicle (bEV) mobility through biological barriers, we use novel multiple-particle tracking technology. The goal is to evaluate changes caused by extracellular vesicles relevant to placental function and neonatal development. Methods/Study Population: We conducted multiple particle tracking to assess whole bacterial and bEV mobility in cervicovaginal mucus. To accomplish this, cervicovaginal mucus was self-collected from 10 women. Mucus samples were characterized via wet mount, Nugent score, and pH measurements. In parallel, we cultured commercially available vaginal bacteria strains in anaerobic conditions. We isolated bEVs via ultracentrifugation, and subsequently characterized them via nanoparticle tracking analysis to measure size, ζ-potential, and concentration. We investigated reproductive tract tissues response to bEVs. We dosed vaginal, endometrial, myometrial, and placental cells lines with bEVs over a 24 h period and determined uptake, viability, and cytokine production. One-way analysis of variance was used for statistical analysis. Results/Anticipated Results: Based on our previous work, size and ζ-potential greatly affect particle mobility in mucus. G. vaginalis and M. mulieris were smaller than L. crispatus and L. iners. G. vaginalis had a more net-neutral ζ-potential compared to other bEVs. During multiple-particle tracking analysis, whole bacteria were unable to diffuse through vaginal mucus, while bEVs showed increased mobility. Through fluorescence levels, we determined M. mulieris bEVs reach >90% uptake at 24 h. Uptake was verified via microscopy. Across all strains, bEVs were not detrimental to placental viability. When investigating cytokine production in placental cells, an increase in IL-6 was seen after treatment with L. iners bEVs, while TNFα was increased after treatment with G. vaginalis bEVs. Discussion/Significance of Impact: Vaginal microbiome dysbiosis increases adverse obstetric indications. We demonstrate that bacteria are unable to ascend to reproductive tissues. We propose that bEVs travel through vaginal mucus, facilitating microbe–host communication. This impacts obstetric disease pathology and is relevant for diagnostic criteria during pregnancy.

Objectives/Goals: Cardiovascular disease, particularly myocardial infarction (MI), is a leading cause of death in the USA. Previous studies have identified CD8+ T-cells as adverse regulators post-MI. We hypothesized that CD8+ T-cells impair cardiac function by altering scar composition. Methods/Study Population: MI was induced by permanent ligation of the coronary artery in C57BL6/J (WT; 3–7 mo) and CD8atm1mak mice (CD8-/-; 3–7 mo). CD8-/- mice were injected with either vehicle or naïve splenic CD8+ T-cells (2x10^6 cells/injection) via tail vein, 4 hours after ligation. Tissue was collected at Day 7 post-MI for biomechanical testing and further downstream analyses. Granzyme (Gzm)A, B, and K were tested for collagen cleavage using a fluorogenic cleavage assay. Effect on collagen production in TGF-β-activated cardiac fibroblasts was assessed in vitro by stimulating cells with GzmA, B, and K (25 AU) for 24 hours. Results/Anticipated Results: CD8-/- mice had improved ejection fraction and LV dilation at Day 7 post-MI compared to WT and CD8-/- mice resupplemented with splenic CD8+ T-cells (p Discussion/Significance of Impact: Our study demonstrates that CD8+ T-cells regulate cardiac fibrosis partially through Gzm release, resulting in left ventricular biomechanical impairments and increased dilation.

Objectives/Goals: This study aims to harmonize quality indicators (QIs) across University of Toronto-affiliated microbiology labs to establish universal benchmarks that enhance performance, patient safety, and health outcomes. Harmonized QIs will enable effective comparisons and enhance the consistency of care. Methods/Study Population: The study employed the Delphi method, a structured and iterative process to build consensus. An expert panel of clinical microbiology trainees, medical microbiologists, trainees, and site leads from five University of Toronto-affiliated microbiology labs was assembled. Initial insights were gathered through surveys and a comprehensive scoping review of the literature. The study involved two rounds of feedback, a SurveyMonkey-based survey, with a defined consensus of 75% agreement among participants. Followed by an implementation survey conducted through REDCap to assess how these QIs were adopted in practice and identify barriers to implementation. Results/Anticipated Results: The study achieved consensus on nine high-impact quality indicators, including blood culture volume and contamination rates, cerebrospinal fluid transport time, and turnaround times for Gram stain results. Blood culture contamination and positivity rates garnered the highest agreement, at 100% and 91%, respectively. While some indicators were widely accepted and implemented, others faced resistance due to feasibility concerns. The study also identified significant variability in the level of adoption across the participating laboratories, pointing to operational challenges and the need for further efforts to address these barriers. Discussion/Significance of Impact: This study highlights the importance of QI harmonization in improving lab services and patient safety. It reveals challenges in standardizing practices but promotes uniformity in QIs, laying the groundwork for better inter-lab collaboration, consistent outcomes, and improvements in microbiology.

Objectives/Goals: Novel therapeutics to control Staphylococcus aureus (S. aureus) infections are needed for people with cystic fibrosis (CF, PwCF). In this study, our objective is to determine if the pharmacologic MEK1/2 inhibitor compound ATR-002 can restrict the growth of S. aureus clinical isolates and modulate infection in a murine model of S. aureus infection. Methods/Study Population: To evaluate the anti-inflammatory effects of ATR-002 on human macrophages, cells were stimulated with TLR2 agonists FSL1 or Pam3CSK4 with a dose range of ATR-002, and secretion of proinflammatory cytokines were measured by ELISA. To determine the direct antibacterial effect of ATR-002, minimum inhibitory concentration (MIC) assays were performed with the community-associated methicillin-resistant S. aureus strain USA300 and 40 S. aureus isolates from PwCF. To validate our results in vivo, mice were provided i.p. treatment with either vehicle, the MEK1/2 inhibitor compound PD0325901 (20 mg/kg), or ATR-002 (10 mg/kg) prior to intranasal infection with 1x10^7 CFU of USA300. Bacterial burdens at 4- and 24-hour post-infection (p.i.) and inflammatory cell recruitment at 24 hours p.i. were quantified. Results/Anticipated Results: Macrophages treated with ATR-002 exhibited a dose-dependent decrease in secretion of proinflammatory cytokines TNF and IL-8 following TLR stimulation. Our studies identified that ATR-002, but not PD0325901 or other MEK1/2 inhibitors, had direct antibacterial effects, and ATR-002 had an MIC range of 8 to above 64 ug/mL on CF S. aureus isolates. In the murine pulmonary infection model, delivery of ATR-002 and PD0325901 significantly prevented infection-induced loss of body mass and decreased neutrophil inflammation. However, when bacterial burdens were quantified 4-hours p.i., only ATR-002 treatment reduced lung bacterial burden compared to vehicle or PD0325901-treated groups. Discussion/Significance of Impact: These results are the first demonstration of the in vivo anti-inflammatory and antibacterial effects of ATR-002. Our results further demonstrate that ATR-002 exhibits direct antibacterial effects across a collection of clinical isolates of S. aureus. Future studies will continue to investigate the therapeutic potential of ATR-002.

Objectives/Goals: People with insulin-treated diabetes face hypoglycemia risk due to imperfect insulin replacement and impaired counterregulation. We identified the dopamine antagonist, metoclopramide, as a potential treatment. Hypothesis: Treatment with metoclopramide will prevent the development of impaired counterregulatory response to hypoglycemia. Methods/Study Population: In a pre-clinical model, diabetes was induced in 10-week-old Sprague-Dawley rats with streptozotocin (STZ, 65 mg/kg IP). Rats were divided into three groups: 1) diabetic controls (STZ+RS, n = 6), 2) recurrent hypoglycemia (STZ+RH, n = 7), and 3) recurrent hypoglycemia + metoclopramide (STZ+RH+MET, 3 mg/kg IP, n = 7). After 3 days, all rats underwent a hyperinsulinemic (50 mU/kg/min) and hypoglycemic (~45 mg/dl) clamp. In the clinical trial, adults with Type 1 diabetes (age 20–60, ≥5 years duration) were enrolled in a phase II, double-blinded, placebo-controlled trial. Awareness status was assessed via Gold score, and subjects maintained drug regimens and underwent two hyperinsulinemic-hypoglycemic clamps (where blood glucose was lowered to 100, 65, 55, and 45 mg/dl) to assess counterregulation. Results/Anticipated Results: In the pre-clinical model, glucose infusion rates (GIR) to maintain hypoglycemia were higher in STZ+RH (27±0.9 mg/kg/min) than STZ+RS (19±0.8 mg/kg/min, p Discussion/Significance of Impact: Metoclopramide improves glucoregulatory, sympathoadrenal, and counterregulatory responses to hypoglycemia in pre-clinical models, suggesting dopaminergic regulation. While clinical data are still blinded, increased epinephrine and growth hormone responses suggest treatment may preserve or restore counterregulation.

Objectives/Goals: The goal of this study is to investigate the peri-metastatic rims of peritoneal lesions to determine features that predict malignancy using both imaging processing techniques and machine learning. This information will subsequently be added to our existing knowledge of peritoneal lesions to improve classification accuracy as benign or malignant. Methods/Study Population: The study population consists of 521 imaged lesions from 163 subjects with cancers of GI-origin with biopsy results as well as the clinical subject information and follow up. All images were obtained during staging laparoscopy by the senior author (TS). On the images, the central lesion as well as the surrounding peri-metastatic rim will be segmented as regions of interest (ROIs). Image processing will be used to calculate a variety of metrics for these two regions. A general estimating equation approach will be used to determine significance of these metrics compared to the dependent outcome of malignancy determined on the pathology report as the ground truth. These ROIs and significant metrics will then be used to improve the accuracy of a machine learning model to classify these lesions as benign or malignant. Results/Anticipated Results: Our previous research showed that experts performed this task at only a 52% accuracy rate (classifying lesions as malignant or benign based on imaging). A previous machine-learning model on a much smaller dataset was able to achieve by contrast an area under the curve of 0.78. We anticipate that by including a larger dataset in addition to including the peri-metastatic rim, we will be able to improve the accuracy of the the model in this task while uncovering significant biomarkers as well that can be used in future studies. Discussion/Significance of Impact: Classifying peritoneal lesions determines the correct treatment for cancer patients whether chemo-radiation, definitive surgery or palliative surgery. This project aims to develop an improved model that can perform this task using nonlabeled laparoscopic imaging with a particular focus on the diagnostic value of the peri-metastatic rim.

Objectives/Goals: • To determine the impact of liraglutide on inflammation and organ injury during sepsis. • To investigate the protective effects of liraglutide on microvascular dysfunction in a clinically relevant model of sepsis. • To provide evidence for the potential therapeutic use of GLP-1 receptor agonists in endothelial dysfunction in sepsis. Methods/Study Population: Sepsis was induced in mice (N = 34) by intraperitoneal injection of cecal contents (1.8 mg/g body weight) and 24-hour hyperoxia (FiO2 90–95%). Mice received saline or liraglutide (0.1 mg/kg) at 6 and 18 hours post-injection and fluids and antibiotics at 12 hours. At 24 hours, mice were euthanized for plasma, bronchoalveolar lavage (BAL), and tissue collection. Plasma inflammatory markers, organ injury markers, and BAL components were measured. In vitro, primary human lung microvascular endothelial cells (HLMVECs) were treated with saline or liraglutide for 24 hours before exposure to saline or LPS (100 ng/mL). HLMVEC barrier dysfunction was evaluated using express permeability testing (XPerT) and electric cell-substrate impedance sensing (ECIS) to measure transendothelial electrical resistance (TER). Results/Anticipated Results: In murine sepsis, illness severity scores and lung injury were improved in mice pretreated with liraglutide (N = 10). Plasma blood urea nitrogen (BUN; P = 0.0036), alanine transaminase (ALT; P = 0.0311) and vascular inflammatory markers MCP-1 (P = 0.0172), ICAM-1 (P = 0.0356), and Pecam-1 (P = 0.0493) in plasma were reduced in mice treated with liraglutide. In HLMVECs, liraglutide (1.5 nM) significantly reduced LPS-induced barrier dysfunction measured by XPerT assay (P = 0.0030) and ECIS (P = 0.0075). Discussion/Significance of Impact: Liraglutide reduces illness severity, vascular inflammation, and organ injury in a two-hit model of sepsis. Liraglutide has direct effects in the microvascular endothelium, limiting LPS-mediated barrier dysfunction. These findings support a protective role for GLP-1 receptor agonism in sepsis, mediated through the microvasculature.

Objectives/Goals: The aging population faces unique health issues, many of which are exacerbated by aging-associated immune function decline. However, the driving mechanisms behind this decline are poorly understood. We use a mouse model to study the relationship between extracellular matrix (ECM) stiffness and cell mobility within the lymph node (LN) as one potential driving mechanism. Methods/Study Population: We will collect LN from young (6 weeks old) mice and acquire LN from aged (11 month old) mice from a collaborator. We will section LN for ex vivo analysis, including quantification and localization of collagen I using immunofluorescent staining, analysis of microrheological properties using multiparticle tracking (MPT) with PEGylated fluorescent nanoparticles, and migration assays to track the movement of B and T cells. Results/Anticipated Results: We hypothesize a positive correlation between collagen deposits and stiffness within murine LNs due to known mechanisms underlying age-related fibrosis. We also hypothesize that areas of increased stiffness (as revealed by MPT) will exhibit decreased cell migration due to physical hindrance to B and T cell mobilization. Furthermore, we hypothesize that aged murine LN will exhibit a significant increase in stiffness and resultant decreased cell mobility when compared to young murine LN, particularly in areas with increased collagen localization. Discussion/Significance of Impact: These studies will elucidate structure–function relationships driving age-associated LN fibrosis and stiffness, and the resultant impedance to cell migration, thus clarifying some of the potential driving mechanisms behind immune aging and providing data capable of informing the development of relevant models and interventions.

Objectives/Goals: The never in mitosis kinase (NEK) family regulates vital processes, namely cell cycle progression, but their potential as therapeutic targets in TNBC has not been fully explored. Our studies aim to develop a toolkit to investigate the functional roles of NEKs in pathologies including carcinogenesis. Methods/Study Population: To assess differential NEK expression in normal and tumor tissues and correlation of gene expression with patient survival, we used Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan–Meier Plotter (KMPlot) pan-cancer analysis, respectively. Basal NEK protein levels were determined by immunoblot across a panel of cell lines, including breast cancer, osteosarcoma, hepatocellular carcinoma, and non-cancerous cells, to identify appropriate systems for evaluation of NEK function. Doxycycline-inducible cell lines were generated by transduction with lentiviral stocks of NEK shRNA and overexpression constructs and antibiotic selection. Expression was analyzed by qPCR and immunoblot. Results/Anticipated Results: Expression of NEK2, 4, 5, 6, 8, and 11 was higher in breast tumors compared to normal tissue by GEPIA analysis. Further examination using KMPlot showed a correlation between elevated NEK6 expression and decreased overall survival in patients with aggressive cancers. As an initial proof-of-concept study, we analyzed NEK6 protein expression in breast cancer cells. Levels of NEK6 were elevated in TNBC cells (MDA-MB-231) compared to hormone receptor positive (HR+) breast cancer cells (MCF7). Using complementary approaches to investigate the functional role of NEK6 in breast cancer, we depleted NEK6 expression using shRNAs in TNBC cells and expressed NEK6 in HR+ cells Discussion/Significance of Impact: Because kinase dysregulation promotes oncogenesis and metastasis, targeting kinases is a key strategy in therapeutic development. A NEK-specific molecular toolkit allows researchers to elucidate NEK functions and contributions to carcinogenesis, promoting advancement of novel therapies.

Objectives/Goals: To develop and deploy an academic learning health system (aLHS) Bridge Program to capitalize on our unique organizational strengths in Implementation Science (IS) and to overcome the gap between science and practice that threatens the success of an aLHS. The aLHS Bridges includes an IS Shared Resource, intended to advance IS to practice. Methods/Study Population: The new IS Shared Resource is built on our expertise in dissemination and implementation science, pragmatic, and adaptive trials and the CTSI’s prior success in integrating academic and clinical missions. We also leveraged our existing experts to co-lead the aLHS Bridge including Kristie Foley, PhD, inaugural Chair of the Department of Implementation Science, and Gary Rosenthal, MD, Chair of the Department of Internal Medicine. Specifically, the new IS Shared Resource builds on the capacity of the Department of IS, comprised of 36 faculty members (19 primary and 17 secondary/adjunct appointments) with expertise in qualitative and mixed-methods research, stakeholder engagement, participatory research, digital health, and organizational theory. Results/Anticipated Results: The IS Shared Resource is primed to aid faculty with dissemination and implementation needs, including shortening the time of intervention adoption and using implementation science to inform sustainable and effective implementation practice. The IS Shared Resource is equipped to provide consultation services to faculty members to understand their specific request and match IS faculty members who are expertly trained in specific strategies or contexts. Discussion/Significance of Impact: Leveraging current resources and our first-of-its-kind Department of Implementation Science, our CTSI was able to stand up the IS Shared Resource to support the goals of the CTSA and our greater institution mission. Using a multidisciplinary approach was essential to the success of the IS Shared Resource.

Objectives/Goals: This study assesses the feasibility of a human-to-swine lung transplant model for the evaluation of bioengineered organs. Given the critical organ shortage, this research explores bioengineered organs as a potential solution by evaluating early lung function, immune responses, and technical aspects to develop a model for bioengineered lungs. Methods/Study Population: The study employs a non-survival human-to-swine left lung transplant model in an immunosuppressed Yorkshire swine. A combination of cobra venom factor pretreated with methylprednisolone and Benadryl with scheduled dosing of tacrolimus and mycophenolate over a 24-hour period will be administered. The transplanted human lung is assessed over a 24-hour post-transplant period, with hourly pulmonary vein gas sampling and lung tissue resections. The proposed model will assess the immunological response of swine to human lung tissue as well as the efficacy of the immunosuppression model. Tissue samples are taken at intervals to evaluate for signs of rejection, cellular damage, and the overall function of the transplanted lung. All tissues are preserved in formaldehyde for subsequent immunohistology evaluation. Results/Anticipated Results: We anticipate a successful non-survival swine transplant model with pulmonary function sustained for the full 24-hour study using our proposed immunosuppression regimen. Initial testing with a standard human lung will lay the groundwork to assess the effectiveness of the human-to-swine transplant model. Hourly pulmonary vein gas analyses and tissue biopsies are expected to show minimal immune rejection, supported by the preoperative immunosuppression regimen. Early data indicate that the swine tolerates both the surgical procedure and immunosuppressive therapy well, with manageable hemodynamic stability. This model is expected to yield critical insights into lung viability and will identify areas for optimization for long-term survival studies to test the efficacy of bioengineered organs. Discussion/Significance of Impact: This non-survival swine model offers valuable insights into the acute-phase immune response and functional viability of human-to-swine lung transplant model. The findings will support the development of long-term survival models that will allow the evaluation of bioengineered organs based solely on their functionality as engineered organs.